Improved swine erysipelas vaccine



Uite

3,083,142 EMPRGVED SWHNE ERYSEPELAS VACCRNE Dennis George Howell,Rickmansworth, Robert Parr,

South Eating, London, and Kathleen Rosemary Heath,

Greenford, England, assignors to Glaxo Group Limited,

a British company No Drawing. Fiied Feb. 25, 1959, Ser. No. 795,322Claims priority, appli ation Great Britain Feb. 27, 1958 7 (Ilaims. (Cl.167--78) This invention is concerned with improvements in or relating tovaccines.

In the administration of vaccines it is often the case that sufiicientvaccine to produce satisfactory immunity against a particular diseasecannot be administered in a single injection. Many vaccines aretherefore administered in two or even three injections at intervals of afew weeks or more.

Particularly in veterinary medicine, and where it is required tovaccinate large numbers of animals, the labour and hence expenseinvolved in giving injections is considerable and to have to administersecond or even third injections of vaccine may represent a substantialincrease in the cost of rearing the animals. This problem isparticularly acute where range animals are concerned since the labour ofrounding up the herds two or three times in the course of a few weeks isoften prohibitive.

It has been suggested that adjuvants could be added to vaccinepreparations for the purpose of delaying the release of antigen into thesystem and so thereby reducing the number of doses necessary to set up asatisfactory antibody response. in particular, it has been proposed topresent the antigenic material in a water-in-oil emulsion, the antigenbeing contained in the disperse aqueous phase and its absorption intothe system so being delayed by the surrounding continuous oil phase.Such preparations have in many instances proved unsatisfactory in use asthey frequently give rise to considerable undesired local reactions atthe site of injection which may impede the desired release of antigen.

We have now found that a vaccine dispersed in certain types ofoil-in-water emulsion hereinafter referred to possess marked advantagesover the hitherto proposed water-in-oil emulsion-based vaccines. Theoil-in-water emulsions thus used for the purpose of the presentinvention are those which can be described as being stabilised andremaining substantially stable even following injec tion into muscletissue. In experiments, we have carried out, we find that manyemulsions, when injected into living muscular tissue, rapidly break downinto separate oil and water phases with the result that the oil phasetends to cause necrosis or other tissue damage, whilst the separatedaqueous phase no longer has the protecting action of the oil intended todelay release of antigen; this phenomenon is noted even with emulsionswhich in vitro would normally be considered as quite stable. For thepurpose of the present invention, therefore, we use emulsions which arestabilised, i.e. so formulated that they remain substantially stable forsome time following injection into animal muscle tissue.

The vaccines prepared according to the invention cause substantiallyless tissue damage than vaccines hitherto prepared with oily adjuvantsand give rise to a more reliable and usually better delayed release ofantigen.

It has also been observed that vaccine preparations according to theinvention are in general easier to inject than many hitherto proposedvaccines based on water-in oil emulsions, being capable of production ina more readily flowing form.

According to the present invention we provide an injectable vaccinepreparation comprising antigenic ma- 3,e83,142 Patented Mar. 26, 1963terial dispersed in a parenterally acceptable stable emulsion of an oildispersed in a continuous aqueous phase.

The term antigenic material is used in this specification to meanmaterial derived from the culture of a pathogenic organism, whichmaterial on introduction into animal blood gives rise to antibodiesserving to protect against or combat infection.

The oil-in-water emulsions according to the invention will generally beprepared with the aid of parenterally acceptable wetting agents whichserve to form and stabilise the desired emulsion.

As is well known, the formation of an oil-in-water emulsion as distinctfrom a water-in-oil emulsion involves suitable choice of wetting agentor agents, having regard to the relative proportions of the oil andwater phases and their exact nature. The consistency of an emulsion andhence its suitability for injection is also dependent on itsconstituents. Thus for the purpose of the present invention it isnecessary so to choose the constituents of the emulsion that anoil-in-water'emulsion (as distinct from a water-in-oil emulsion) isformed and that its con-- sistency is such that it can be injected. itis further necessary that the emulsion be stable and remain as anemulsion after injection into muscle tissue.

The wetting agents used in the preparation of the stabilised emulsionforming the base of the vaccines according to the invention arepreferably of the non-ionic type. With such wetting agents more reliablestability of the emulsion on injection is secured and there is lesstendency for the wetting agent to be precipitated from I the aqueousphase by components of the antigen preparationi than when a cationic oranionic wetting agent is use We have found it preferable to employ atwo-component wetting agent system, one of the wetting agents servingprimarily to form the emulsion and the second primarily to stabilise theemulsion once formed. As is known, for example, the Water-soluble orpreferentially water soluble wetting agents generally act as emulsifyingagents whilst wetting or surface active agents which are oil-soluble (orpreferentially oil soluble) are known generally to exert a stabilisingeffect on emulsions and many different wetting agents are now oifered onthe market both for emulsifying and stabilising purposes.

The following types of Wetting agents have been found to be particularlyuseful for the purpose of the present invention.

Type (a).Polyhydric alcohols, or their anhydrides, esterified with fattyacids, for example, sorbitan monostearate, propylene glycolrnonostearate, diglycol monostearate, mannitan mono-oleate, sorbitansesquioleate, glyceryl monostearate, propylene glycol mono-oleate,propylene glycol monolaurate, diglycol monolaurate, diglycolmono-oleate, diglycol monopalmitate, diglycol monoricinoleate,pentaerythritol monostearate, pentaerythritol distearate,pentaerythritol dioleate, pentaerythritol glycerol oleate,pentaerythritol dioleostearate, low-molecular polyoxyethylene oleate,glyceryl mono-groundnut acid ester, polyglyceryl monostearate,polyglyceryloleate, glycol monostearate, glyceryl mono-oleate, glycerylmonopalmitate and low-molecular polyoxyethylene monostearate.

The wetting agents of the above type (a) are, in general, oil-solubleand hence suitable as stabilising agents. The following type, type (b),however, are generally Water-soluble and so suitable as emulsifyingagents.

Type (b).Polyoxyalkylene derivatives of esters of polyhydric alcohols,for example polyoxyethylene sorbitan monostearate, pol'yoxyethylenepropylene glycol monostearate, polyoxyethylene glycol monolaurate,polyoxyethylene glycol mono-oleate, polyoxyethylene glycol monostearate,polyoxyethylene glyceryl stearate, polyoxyethylene sorbitanmonopalmitate, polyoxyethylene sorbitan monolaurate, polyoxyethylenesorbitan trioleate, and polyoxyethylene sorbitan mono-oleate.

Further types of wetting agents which may be of use include thefollowing:

Type (c).Water soluble fatty acid esters of polyethylene glycol, forexample polyethylene glycol 300 dilaurate, polyethylene glycol 300monostearate, polyethylene glycol distearate and nonaethylene glycolmono-oleate, lower molecular polyglycol ricinoleate, lower molecularpolyglycol monocaprate-caprylate, lower molecular polyglycolmonolaurate.

Type (d).Fatty alcohols condensed with ethylene oxide e.g. the materialsold as Collone A.C. or Abracol S.W. These wetting agents arewater-soluble and hence suitable as stabilising agents.

The stabilising agents of type (a) are preferably used in conjunctionwith the emulsifying agents of type (b). Thus, one especially suitablecombination is sorbitan monostearate with polyoxyethylene sorbitanmonostearate while another is propylene glycol monostearate withpolyoxyethylene monostearate, advantageously combined with diglycolmonostearate. The material Collone A.C. is preferably used inconjunction with sorbitan monostearate and/or low-molecularpolyoxyethylene monostearate.

The exact proportion of wetting agents to be used will depend on theirnature. However, using a combination of non-ionic emulsifying andstabilising agents, we find in general that from 0.5 to 1.5% ofemulsifying agent in combination with from 0.5 to 1.5% of stabilisingagent gives satisfactory results. The ratio of emulsifying agent tostabilising agent can thus vary between 1:3 and 3:1. It should be notedthat too great a proportion of Wetting agenteither of the emulsifying orthe stabilising typeis undesirable and may cause instability or evenbreaking of the emulsion or phase reversal.

The proportion of oil to water is important and preferably theproportion of oil to water in the emulsion should not be more than 65%but preferably not less than 30%. Desirably the proportion of oil towater should be within the range of from 40-60%. The composition of theemulsion base should, however, be correlated with the quantity of water(if any) added with the antigen and the final product, in order to bereliably stabilised, preferably contains more than of oil.

In order to determine the suitability of a given set of emulsioncomponents for preparing a stable oil-in-water emulsion of suitablethinness for injection one merely blends the components in a homogeniserand leaves the emulsion so formed to stand for a prolonged period. Anytendency to separation or complete cracking can then be simply observedlWhere there is doubt as to whether an oil-in-water or a Water-in-oilemulsion has been formed, one can add a dye which is soluble in only onephase and examine to see whether the disperse or the continuous phase iscoloured. Alternatively one can determine the conductivity of theemulsion which would be far lower for a water-in-oil emulsion than foran oil-in-water emulsion. The emulsion should be tested in vivo; forexample, it may be injected into the muscle tissue of an experimentalanimal e.g. a mouse, and, after some days, the animal sacrificed and thetissue surrounding the site of injection examined. It the emulsion issufliciently stabilised there should be no substantial necrosis andusually there will be some of the emulsion still present.

The oils which may be employed to prepare the oilin-water emulsions maycomprise vegetable oils, for example arachis, olive, sesame, soya bean,castor and cottonseed oils as well as oily esters e.g. ethyl oleate,dibutyl sebacate and isoamyl salicylate. However, we find that mineraloils of a pharmaceutical grade e.g. light liquid parafiin, are to bepreferred as they in general, and surprisingly, cause even less tissueirritation than the vegetable oils. This we believe to be due topossible greater stability of mineral oil emulsions and hence lesstendency for them to break after injection, the fine oil droplets beingeventually dispersed by phagocytosis.

In the preparation of the vaccines according to the invention it isconvenient to prepare the oil-in-water emulsion by, for example,blending the oil, water and wetting agents in a homogeniser often atelevated temperature, for example at about 60 C., and to mix thisemulsion with the antigenic material in the desired proportions.

The final vaccine must not contain unwanted organisms and it is thusnecessary to sterilise the various components of the vaccine. Where heatis used for sterilisation, the emulsion or other components must besterilised in the absence of the antigenic material, which would bedestroyed by the high temperature. If the emulsion does not withstandheat sterilisation it is possible to sterilise the oily and aqueouscomponents before emulsification. In general, the oil may be sterilisedby heating to about 160 C. for one hour while the aqueous medium may besterilised in the autoclave at 15 lbs. pressure for 30 minutes. Theemulsion should be cooled before mixing with the antigenic materialsince few antigens can withstand temperatures in excess of 40 C. and arebest kept at room temperature or below. However, it is often preferableto emulsify the oil with an aqueous suspension of the antigenicmaterial, particularly where a high concentration of antigenic mate-rialin the final vaccine is required. Thus if the antigen is obtainable onlyin the form of an aqueous suspension and the emulsion is preparedseparately, it is necessary to add a substantial quantity of aqueoussuspension to the emulsion to obtain the required high antigenconcentration which may seriously affect the stability of the emulsion.In such cases it is often possible to avoid the elevated temperaturesgenerally used in preparing the emulsions since, when a Water solublewetting agent, such as polyoxyethylene monostearate, is added .to theantigen suspension and a more oil soluble wetting agent, such aspropylene glycol monostearate, to the oil, it is generally possible toprepare the emulsion by blending at room temperature.

It is also possible to prepare the oil-in-water emulsions from a gel ofthe oil. The gelling agent is usually a metal soap of a long chain fattyacid e.g. aluminium stearate, aluminium oleate, aluminium pal-mitate,lithium stearate, calcium oleate etc. In general it is possible to use asomewhat lower proporation of oil in the emulsion when a gel is used.One suitable gel comprises arachis oil containing between 1.5 and 2.5%aluminium stearate.

While the antigenic material incorporated in the preparations accordingto the invention may be of substantially any kind or in any form, thefollowing have been found particularly suitable for presentation withoil-inwater emulsions.

(1) Bacterial toxoids from which dead bacteria have been removed, forexample, toxoids, from Staphylococcus aureus. Clos-tridial toxoids, inparticular toxoids from Cl. weichii, types B, C and D, which giveprotection in various combinations against lamb dysentry and pulpykidney disease and enterotoxaemia in sheep and toxoids from Cl. tetaniwhich give protection against tetanus.

(2) Bacterial vaccines containing dead bacteria, for examplepreparations from Erysipelothrix rhusiopathiae which give protectionagainst erysipelas in pigs and turkeys, also Brncella, Salmonella andLeptospiral vaccines.

(3)Viral vaccines, for example, Swine fever virus, Foot and Mouth virus,Loupin ill virus, Enzootic abortion virus, infections Larynys tracheitisvirus, Newcastle disease virus, and Infectious Bronchitis virus.

It will be appreciated that it is often advantageous to administer to ananimal two vaccines giving protection against different diseases andthat it is convenient to combine these into a single vaccine forsimultaneous administration. Thus the toxoid from Cl. welchii type Dtoxin, which protects against pulpy kidney disease in lambs, can becombined with toxoid from Cl. tetani, which protects against tetanus, soas to protect young lambs against both these diseases.

The antigenic material may be incorporated into the oil-in-wateremulsion in substantially any convenient form. It may thus be a driedsolid, if desired purified, an adsorbate on a parenterally acceptableadsorban-t, for example aluminium phosphate, or aluminium hydroxide,pumice or kieselguhr or a suspension in a parenterally acceptableliquid. The final vaccine should, however, be pyrogen free and shouldcontain as little material as possible which gives rise to unwantedreaction in the body. The aqueous phase of the final vaccine is alsopreferably substantially isotonic, that is the sodium chlorideconcentration in the aqueous phase is preferably about 0.85%, and it maycontain preservatives such as phenol or thiomersalate.

The invention will now be described with reference to two particularembodiments thereof. It will be understood that these embodiments whilstrepresenting vaccines which are of considerable use are only examples ofmany which can be prepared according to the invention.

VACCINE FOR THE CONTROL OF ERYSIPELAS 1N SWINE Erysipelas in swine iscaused by the organism Erysipelothrix rhusiopathae and hitherto vaccinesfor the immunisation of swine against the disease have been prepared byculturing the organism on suitable nutrient media, killing the organismand adsorbing the immunising antigens so obtained on a suitableadsorbent, e.g. alumina, which is then suitably formulated forinjection. The adsorbate of antigens has previously been proposed to beadministered in various media, the principal use in Great Britain havingbeen of simple aqueous suspensions; such preparations have usually beenof unsatisfactory potency, and have given only relatively shortimmunity. To overcome these diificulties a vaccine has been proposedwhich is in the form of a water-in-oil emulsion but it was notacceptable due to the severe local reactions which occurred afterinjection and was, furthermore, diificult to inject.

We have found that a particularly effective vaccine preparation for thecontrol of erysipelas in swine can be formulated comprising a stabilisedemulsion of a parenterally acceptable oil in water, having dispersedtherein an adsorbate of immunising antigenic material derived from theculture of Erysipelothrix rhusiopathae upon an injectable adsorbent.Such vaccines in accordance with the present invention can readily beprepared and have good antigenicity and are stable, readily injectable,do not, in general, give rise to marked reactions and give improvedprolongation of action as compared with simple aqueous suspensions of anadsorbate of the antigens. In particular they are surprisingly betterfrom various points of view, especially ease of injection, reducedtendency to cause reactions, etc., than erysipelas vaccines based onwater-in-oil emulsions previously proposed.

In the preparation of the vaccines according to the invention, it ispreferred to prepare separately an adsorbate of the antigenic materialand blend this with an emulsion of the chosen oil in water; preferablythe adsorbate of the antigens is first suspended in water containing abactericidal agent, e.g. formalin or thiomersalate, and made isotonicwith blood e.g. with the addition of sodium chloride. This suspension isthen blended with the emulsion.

Preferably the proportion of oil to water in the emulsion beforeaddition of the antigenic material is not more than 65% and isadvantageously within the range of from 4060%.

The proportion of adsorbate is also preferably within the range of from5 25 conveniently The adsorbate of the antigens may be formed from anysuitable adsorbent, examples of which are aluminum hydroxide, aluminumphosphate, pumice and kieselguhr. The adsorbate may be prepared in anydesired way, for example by culturing the organism on a suitablenutrient medium, inactivating the culture containing antigens with forexample formalin or thiomersalate, adding adsorbent to adsorb theantigens, the resultant adsorbate being separated, for example bycentrifugation. 1

A virulent strain of organism should naturally be used for theproduction of this vaccine and, in general, organisms showing nosubstantial filamentous growth are suitable. We have found it preferableto culture the or ganism at or about 30 C., instead of the more usual 37C., as in this way a more virulent culture is obtained. The medium usedfor the culture organism may be of standard type.

VACCINE FOR THE CONTROY OF LBPTOSPIROSIS IN CATTLE AND OTHER ANIMALSLeptospirosis can be caused by the organism Laptospira pomonai, thedisease occurring in two phases. In the first phase, which is the acutephase, the infected animal shows clinical manifestations of the diseasewith accompanying fever, haemoglobinuria, and loss of condition whichsometimes results in death. In the second phase the leptospira organismscolonize the kidney tubules and the animals, although apparently freefrom infection, become carriers, shedding viable leptospira organisms inthe urine. They are thus a source of infection to any susceptible animalin the vicinity.

We have found it possible to formulate a particularly useful vaccineactive against leptospirosis comprising a stabilised parenterallyacceptable oil-in-water emulsion containing antigenic material derivedfrom the culture of Leptospira pomona.

We find that by formulating the new vaccine in a stabilised oil-in-wateremulsion a product is obtained which gives good protection against bothphases of leptospirosis and has reasonably prolonged action. Thus ournew vaccine is more reliable and effective than leptospirosis vaccinesformulated in various other vehicles. In particular the new vaccine ismore acceptable than a similar product in the form of a water-in-oilemulsion.

The vaccine according to the invention is prepared by culturingLeptospira omona, preferably a virulent strain thereof, in a culturemedia therefor, killing the organism and incorporating the antigenicmaterial thereby obtained into a stabilised oil-in-water emulsionaccording to this invention. Preferably the whole culture broth is usedin the formulation of the vaccine.

We have found further that the choice of means used to kill the organismis of importance if best results are to be obtained. Thus, whilst forexample thiomersalate, formalin or heat may be used for this purpose,much improved results are obtained by the use of a phenol. Such phenolsinclude phenol itself, as well as the cresols resorcinol and thexylenols, of which phenol is preferred.

Various media may be used to support the growth of Leptospira pomona aswill be well known to those skilled in the art. As with allmicroorganisms the cultural requirements of Leptospira pomonaessentially comprise a source of nitrogen, a source of carbon and energyand nutrient salts. Very many materials are now known for this purpose,so that the choice of nutrients is very wide and it is a simple matterto choose nutrients suitable for the culture of Leptospira pomona. Weprefer to use Korthofs medium (Leptospirosis in Man and Animals byAlston and Broom published by F. & S. Livingstone, London, 1958, page303) containing sheep serum but other media which can be used includeVervoorts medium as modified by Schuifner (ibid., page 302) and Stuartsmedium (ibid., page 304).

The culture is preferably carried out in at least two and preferablythree stages. For example in the first or primary stage a culture ismade from a suitable source of a,osa,142

the organism e.-g. an infected animal and grown up to form a suitableinoculum for a secondary or development stage. The culture from thesecondary stage is then used as inoculum for a production stage. Themedia used are conveniently of the same constitution for each stage.

A suitable temperature for the culture is 27C.

Following the culture the organims are killed and the broth is thensuitable for formulation.

In the formation of the vaccine we prefer first to form an oil-in-Wateremulsion base and then incorporate the antigenic material therein carebeing taken to keep the emulsion as an oil-in-water emulsion.

Preferably the proportion of oil to water in the emulsion and beforeaddition of the antigenic material is not more than 65% and isadvantageously within the range of from 40-60%.

After formation of the emulsion base, the antigenic material preferablyas the whole culture broth is incorporated therein by any convenientmethod, preferably by use of a high speed blender or homogeniser. Theproportion of culture broth to be incorporated in the emulsion base ispreferably from 40 to 60% of the base.

If desired instead of first forming an emulsion base and thenincorporating the anhydrous material therein, the culture broth may forexample be simply diluted as necessary and the oil emulsified therein.In this case it is preferable to add a water-soluble wetting agent tothe aqueous suspension and an oil-soluble agent to the oil.

The use of vaccine adjuvants, such. as aluminum hydroxide gel oraluminium phosphate is not necessary according to the invention, butsuch adjuvants may if desired be used. Thus for example the antigenicmaterial may be adsorbed on to some suitable adsorbent and theadsorbates incorporated into the emulsion base. The direct incorporationof the whole culture broth into the base i however preferred.

For the better understanding of the invention we now give the followingexamples by way of illustration only.

Example 1.Preparation of Swine Erysipelas Vaccine (a) PREPARATION OF THECULTURE BROTH A liver extract is first prepared by infusing 9 lbs. ofminced fat free, fresh ox liver with 9,200 mls. of distilled water,overnight in a refrigerator (+4 C.). This is then steamed for 2 hours(100 C.) and filtered through muslin and then Whatmans No. 54 filterpaper. The filtrate is then sterilised in an. autoclave at 10 lbs.pressure for 20 minutes for storage purposes.

The culture is then prepared'from:

Liver extract (as above) 8%.

NZ. amine (type E) 4% in distilled water. Sodium chloride 0.5%.

Glucose 0.05%.

Heat to dissolve and adjust the pH to 7.5. Filter while still hotthrough Whatmans No. 54 filter paper. Sterilise by autoclaving at 10lbs. pressure for 30 minutes. Add sterile inactivated horse serum, 50mls. per litre. Incubate at 37 C. to test for sterility.

(1)) THE PREPARATION OF ALUMINIUM HYDROXII DE.

This is prepared by the method described in the World HealthOrganisation report No. 61. The gel is precipitated from aluminiumsulphate by ammonia in the presence of ammonium sulphate, washed freefrom soluble salts and sterilised by autoclaving.

The aluminium hydroxide content is calculated to the weight in grams perml. of gel.

(0) OIL-IN-VVATER EMULSION This is a sterile liquid parafiin emulsionsyringeable through a No. 26 (British standard wire gauge) needleprepared from: 7

Light liquid parafiin (medicinal ride and thiomersalate in the water,then sterilise at 15 lbs. pressure for 30 minutes.

Add the water to the oil when both phases are at a temperature of 60 C.Homogenise. The final product contains of this emulsion.

(d) VACCINE PREPARATION Culture and incubati0n.-'Ihe strain employed forvaccine production is maintained in a freeze dried state (lyophilised)and when required is reconstituted in liver serum broth medium,incubated overnight at 30 C. and tested for purity. This culture is thenused to inoculate the amount of broth required as an inoculum for thevaccine. 0

An 18 hour liver serium broth culture is used, adding 10 mls. ofinoculum to 1 litre of medium. This is incubated at 30 C. for 48 hours.

Killing of the bacteria.Add thiomersalate to the culture to give a finaldilution of 0.02% or formalin 0.25%. Leave the culture in the incubatorat 30 C. for 3 days and then test for any live bacteria by adding aquantity of the culture to a digest meat broth containing 0.1% cysteinehydrochloride to counteract the presence of any thiomersalate.

Adsorption.--When the culture is proved to be completely dead, enoughaluminium hydroxide gel is added to give a final concentration of 1 mgm.per ml. The vaccine is shaken well and left overnight at +4 C. toadsorb.

Concentration.The vaccine is now put through a centrifuge and thesupernatant fluid removed. The deposit is scraped from the bowl of thecentrifuge and thiornersalate saline is added. (The deposit from litresof culture is made up to 2 litres with thiomersalate saline. Thisconstituted 10% of the final vaccine.) A mechanical stirrer is used tobreak up the deposit, which is then added to the emulsion andhomogenised. The deposit, from 100 litres of culture being made up to 20litres with the emulsion so concentrating five times.

Example 2.-Preparati0n of Leptospirosis Vaccine Distilled water to 1litre.

The medium is boiled to dissolve the constituents, autoclaved to allowany phosphate to precipitate and filtered. The pH is adjusted to 7.2 andthe medium is bottled and autoclaved.

Before inoculation 10% sheep serum deactivated at 56 C. for two hours isadded to the medium.

CULTIVATION OF VACCINE Primary cultures.-To medium containing serum isadded 0.5 ml. of blood from a heavily infected hamster. The primaryculture is incubated for seven days. Checks are made on sterility andthe leptospira is typed against standard immune sera.

Secondary cultures-5.0 ml. of actively growing primary culture is addedto 150 ml. conical flasks containing 50 ml. of the above medium and 5ml. of serum. These flasks are incubated to maximum growth which isgenerally achieved in from seven to fourteen days.

Final vaccine cultures-The leptospira are grown in 1 litre flaskscontaining 500 ml. of medium and 50 ml. of serum. One secondary cultureis added to each vaccine flask and the whole incubated for seven days.Tests for sterility are carried out on the fifth day. The cultures inall the flasks are pooled after seven days growth. An aliquot is removedand counted. Phenol to 0.5% is then added to kill the leptospira and thepooled vaccine is refrigerated at 4 C.

After seven days the vaccine is then mixed in 50:50 proportions with anoil-in-water emulsion prepared as follows:

OIL AND WATER EMULSION This a sterile liquid paraffin emulsionsyringeable through a No. 26 (British standard wire gauge) needleprepared from:

Light liquid paraffin (medicinal grade) 44.5% W./v. Grill 8 (emulsifyingagent) 1.1% W./V. Crill 3 (stabilising agent) 0.9% w./v. Sodium chloride0.85 Vehicle Thiomersalate 0.015% to 1007 Distilled water to 100%Sterilise the parafim by heating at 160 C. for one hour. Disperse thecrills and dissolve the sodium chlo ride and thiomersalate in the Water,then sterilise at 15 lbs. pressure for 30 minutes.

Add the Water to the oil when both phases are at a temperature of 60 C.Homogenise.

Further emulsion bases are illustrated in the following examples.

In this example the light liquid paraffin may be replaced with an equalamount of arachis oil.

Example Sorbitan monostearate (stabilising agent) Polyoxyethylenesorbitan monostearate (emulsifying agent) 1.0 Aluminium stearate/arachisoil gel (2.4%) 35.0 Saline 62.0

We claim:

1. An injectible vaccine preparation in the form of an oil-in-wateremulsion comprising a preformed emulsion base which is stable in thepresence of body fluids upon 5 intramuscular injection and is anoil-in-Water emulsion containing an aqueous phase substantially isotonicto the blood and an oil phase consisting essentially of from to 65% ofmineral oil, from 0.5 to 1.5% of a watersoluble nonionic emulsifyingagent and from 0.5% to 1.5 of an oil-soluble nonionic stabilising agent;and an antigenic material consisting of dead Erysipelothrixrhusiopaflzol, the entire preparation containing more than 10% ofmineral oil.

2. A preparation as claimed in claim 1 in which said antigenic materialis an adsorbate on a parenterally acceptable adsorbent. 3. A preparationas claimed in claim 1 in which said emulsifying agent is apolyoxyalkylene derivative of a hexitan ester and said stabilising agentis a fatty acid ester of a polyhydric alcohol.

4. A preparation is claimed in claim 1 in which said emulsifying agentis polyoxyethylene sorbitan monost-earate and said stabilising agent issorbitan monostearate.

5. A preparation as claimed in claim 1 in which the proportion of oil towater in the emulsion base is between 40 to 60%.

6. A preparation as claimed in claim 1 in which said oil includes agelling agent.

7. A preparation as claimed in claim 6 in which said gelling agent is ametal soap of a long chain fatty acid.

References Cited in the file of this patent UNITED STATES PATENTS2,057,623 Beard Oct. 13, 1936 2,529,461 Schneiderwirth Nov. 7, 19502,675,343 Clymer Apr. 13, 1954 2,756,176 Maurer et a1 July 24, 1956FOREIGN PATENTS 425,406 Great Britain Mar. 7, 1935 OTHER REFERENCES

1. AN INJECTIBLE VACCINE PREPARATION IN THE FORM OF AN OIL-IN-WATEREMULSION COMPRISING A PREFORMED EMULSION BASE WHICH IS STABLE IN THEPRESENCE OF BODY FLUIDS UPON INTRAMUSCULAR INJECTION AND IS ANOIL-IN-WATER EMULSION CONTAINING AN AQUEOUS PHASE SUBSTANTIALLY ISOTONICTO THE BLOOD AND AN OIL PHASE CONSISTING ESSENTIALLY OF FROM 30% TO 65%OF MINERAL OIL, FROM 0.5% TO 1.5% OF A WATERSOLUBLE NONIONIC EMULSIFYINGAGENT AND FROM 0.5% TO 1.5% OF AN OIL-SOLUBLE NONIONIC STABILISINGAGENT; AND AN ANTIGENIC MATERIAL CONSISTING OF DEAD ERYSIPELOTHRIXRHUSIOPATHOL, THE ENTIRE PREPARATION CONTAINING MORE THAN 10% OF MINERALOIL.